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WO2023141829A1 - Procédé pour effectuer simultanément un séquençage d'adn de génome entier et une méthylation d'adn de génome entier et/ou un séquençage d'hydroxyméthylation - Google Patents

Procédé pour effectuer simultanément un séquençage d'adn de génome entier et une méthylation d'adn de génome entier et/ou un séquençage d'hydroxyméthylation Download PDF

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WO2023141829A1
WO2023141829A1 PCT/CN2022/074093 CN2022074093W WO2023141829A1 WO 2023141829 A1 WO2023141829 A1 WO 2023141829A1 CN 2022074093 W CN2022074093 W CN 2022074093W WO 2023141829 A1 WO2023141829 A1 WO 2023141829A1
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sequencing
dna
strand
methylation
nascent
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Chinese (zh)
Inventor
杨林
夏军
陈恬
张艳艳
陈芳
聂自豪
张韶红
杨贵芳
王业钦
吕硕
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to PCT/CN2022/074093 priority Critical patent/WO2023141829A1/fr
Priority to CN202280052323.8A priority patent/CN118076734A/zh
Publication of WO2023141829A1 publication Critical patent/WO2023141829A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for simultaneously performing whole-genome DNA sequencing and whole-genome DNA methylation or/and hydroxymethylation sequencing.
  • DNA methylation is an epigenetic regulatory modification that participates in the regulation of protein synthesis without changing the base sequence.
  • DNA methylation is a very wonderful chemical modification. The care of loved ones, the aging of the body, smoking, alcoholism and even obesity will be faithfully recorded on the genome by methylation. The genome is like a diary, and the methylation is used as words to record the experience of the human body.
  • DNA methylation is an important epigenetic mark information. In mammals, the most common methylation modification occurs on cytosine, mainly including 5-methylation modification (5mc) and 5-hydroxymethylation modification (5hmc), obtaining genome-wide methylation level data of all cytosines is of great significance for the study of spatiotemporal specificity of epigenetics.
  • mapping of DNA methylation levels across the genome and the analysis of high-precision methylation modification patterns in specific species will surely have milestone significance in epigenomics research , and lay the foundation for the research of basic mechanisms such as cell differentiation and tissue development, as well as animal and plant breeding, human health and disease research.
  • Whole Genome Bisulfite Sequencing WGBS Whole Genome Bisulfite Sequencing
  • Whole Genome Bisulfite Sequencing that is, whole genome bisulfite sequencing
  • the premise of methylation sequencing is that the whole genome DNA information of the species has been obtained. After bisulfite treatment, the methylated C remains unchanged, and no The methylated C is converted to U, and the methylation sequencing results are compared with the genome information to obtain the modification of cytosine at this position; 2.
  • the unmethylated C base after bisulfite treatment will be converted into a U base
  • the GC content of the whole genome changes drastically, resulting in great amplification and sequencing bias in the subsequent amplification; 3.
  • C cytosine
  • T thymine
  • the comparison (map) of the results obtained by sequencing to the reference genome is less efficient, and there will be too many multiple alignments , leading to abnormal alignment, and even if the sequencing throughput is increased in some positions, effective DNA methylation information cannot be obtained, resulting in the loss of gene-wide methylation information.
  • the present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent. For this reason, the present invention proposes linker element, linker element composition, test kit and its application, the construction method of sequencing library, sequencing library and its application in sequencing and carry out whole-genome DNA sequencing and whole-genome DNA methylation simultaneously Or/and a method for sequencing hydroxymethylation, using the sequencing library for sequencing, a method and system that can simultaneously sequence whole genome DNA and DNA methylation or/and hydroxymethylation sequencing, and DNA and DNA Simultaneous sequencing of methylation or/and hydroxymethylation sequencing is done on one molecule, which can accurately obtain methylation information without reference to gene information, and can accurately locate the methylation position, greatly improving methylation Accuracy of methylation or/and hydroxymethylation sequencing information.
  • the invention proposes a joint element.
  • the linker element is a bubble-shaped single-stranded nucleic acid
  • the single-stranded nucleic acid has a non-complementary region and a complementary region formed by a 5' end sequence and a 3' end sequence, and the 5' end Or have a cohesive end at the 3' end.
  • the positive and negative strands can be effectively connected to form a circular DNA molecule for subsequent DNB (DNA nanoball) preparation experiments.
  • the above joint element may also have the following additional technical features:
  • the cohesive end or the complementary region has an endonuclease recognition site.
  • a cut is formed, and the chain is extended at the cut to obtain a nascent chain.
  • the base at the cohesive end is a U base or a T base.
  • the cohesive end is a U base, it can be used as an endonuclease recognition site for digestion with User endonuclease.
  • the endonuclease is selected from USER endonuclease, DNase endonuclease, RNase endonuclease.
  • the endonuclease recognition site is selected from U bases, deoxynucleotides or ribonucleotides.
  • the adapter element contains one or more sequencing primer sequences, molecular tag sequences and/or sample tag sequences.
  • the length of the joint element is 20-200 nt.
  • the positive and negative strands can be effectively connected to form a circular DNA molecule for subsequent DNB preparation experiments.
  • the linker elements are deoxyribonucleotides and/or ribonucleotides.
  • the linker element has a nucleotide sequence as shown in SEQ ID NO: 1 or 2 or a nucleotide sequence having at least 80% homology therewith.
  • the present invention provides a joint element composition.
  • the linker element composition includes two aforementioned linker elements, and at least one of the linker elements has an endonuclease recognition site on its sticky end or complementary region.
  • the positive and negative strands can be effectively connected by using the adapter element composition according to the embodiment of the present invention to form a circular DNA molecule for subsequent DNB (DNA nanoball) preparation experiments.
  • the linker element composition includes: linker element 1, the linker element 1 has a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleoside having at least 80% homology therewith acid sequence; linker element 2, said linker element 2 has a nucleotide sequence as shown in SEQ ID NO: 2 or a nucleotide sequence with at least 80% homology therewith.
  • the present invention provides a kit.
  • the kit includes: the aforementioned linker element and the linker element composition.
  • the present invention proposes the application of the aforementioned linker elements, linker element compositions, and kits in the construction of sequencing libraries.
  • the sequencing library is used for at least one of whole-genome DNA methylation sequencing and hydroxymethylation sequencing and whole-genome DNA sequencing.
  • methylation or/and hydroxymethylation information can be accurately obtained using the aforementioned linker elements.
  • the present invention proposes a method for constructing a sequencing library. According to an embodiment of the present invention, the method includes:
  • the linker element 1 is selected from the aforementioned linker elements, and the sticky end or the complementary region has an endonuclease recognition site;
  • the cytosines in the nascent chain are all methylated modified cytosines or are all unmethylated modified cytosines;
  • dumbbell-shaped double-stranded DNA the sequence of the nascent strand remains unchanged, and the unmethylated cytosine on the template strand will be converted into uracil or the methylated and/or Hydroxymethylated cytosines are converted to dihydrouracils, resulting in sequencing libraries.
  • the two positive and negative strands of a DNA molecule are connected through the linker element 1, and a nick is formed on its endonuclease recognition site, so that chain extension can be performed on the nick to generate a new chain.
  • a closed DNA loop can be formed to obtain a dumbbell-shaped double-stranded DNA, which is helpful for the subsequent preparation of DNA nanospheres. Sequencing libraries are obtained by converting dumbbell-shaped double-stranded DNA so that all uracils are converted to dihydrouracils.
  • the whole genome sequence can be obtained based on the sequence information of the nascent strand, and the methylation/hydroxymethylation information can be accurately obtained by comparing the whole genome sequence with the sequence information of the template strand.
  • the simultaneous sequencing of DNA and DNA methylation or/and hydroxymethylation is completed on one molecule, and methylation information can be accurately obtained without reference to gene information, and the methylation position can be precisely positioned, greatly improving Improve the accuracy of methylation information.
  • the above-mentioned method for constructing a sequencing library may also have the following additional technical features:
  • the fragmentation is to randomly interrupt or cut double-stranded DNA by physical or chemical methods.
  • the fragmentation is performed by physical ultrasonic method or enzyme reaction method.
  • the blunt end repair is performed using T4 DNA polymerase or mung bean nuclease.
  • T4 DNA polymerase or mung bean nuclease.
  • the phosphorylation is performed by nucleotide kinase.
  • the phosphorylation is performed by using T4 polynucleotide kinase (T4 DNA phosphokinase).
  • the addition of base A at the 3' end is performed by using rTaq enzyme or Klenow polymerase without 3-5 exonuclease activity.
  • rTaq enzyme or Klenow polymerase without 3-5 exonuclease activity.
  • the base of the sticky end is selected from U base or T base;
  • the endonuclease is selected from USER endonuclease, DNase endonuclease or RNase endonuclease;
  • the enzyme recognition site is selected from U base, deoxyribonucleic acid or ribonucleic acid, and the number of said cuts is one or more.
  • the extension uses a DNA polymerase with 5-3 exonuclease or 5-3 displacement function.
  • the DNA polymerase is selected from T4 DNA polymerase, phi29 DNA polymerase or Bst DNA polymerase.
  • T4 DNA polymerase phi29 DNA polymerase
  • Bst DNA polymerase phi29 DNA polymerase
  • all cytosines in the dNTPs used in the extension are methylated or all unmethylated cytosines. Due to methylated cytosine, after bisulfite conversion treatment, the sequence remains unchanged, or with unmethylated cytosine, after conversion treatment (such as using TET enzyme, potassium perruthenate, beta Glycosyltransferase and TET enzyme are converted), the sequence remains unchanged, and genomic DNA information can be obtained by sequencing it.
  • the cytosines in the nascent strands are all methylated cytosines
  • step 6) includes: subjecting the dumbbell-shaped double-stranded DNA to bisulfite treatment to obtain a sequencing library.
  • all the cytosines on the nascent chain are methylated, and after the bisulfite in step 6), its sequence remains unchanged, and it is sequenced Genomic DNA information is available.
  • the unmethylated cytosine will be converted into uracil, which is sequenced, and the sequencing result is compared with the genomic DNA information obtained above to know Methylation information.
  • the cytosines in the nascent strands are all methylated cytosines
  • step 6) includes: converting the dumbbell-shaped double-stranded DNA to obtain a sequencing library, and the conversion process uses
  • the reagent comprises: auxiliary reagent and pyridine borane or bisulfite;
  • the auxiliary reagent is selected from one of the following three: TET enzyme; potassium perruthenate; beta glycosyltransferase and TET enzyme; the conversion
  • the treatment includes: sequentially treating the dumbbell-shaped double-stranded DNA with auxiliary reagents and pyridine borane or treating the dumbbell-shaped double-stranded DNA with bisulfite.
  • TET enzyme recognition can recognize 5mc and 5hmc
  • beta glycosyltransferase can recognize 5mc
  • potassium perruthenate can recognize 5hmc.
  • cytosines on the nascent chain are unmethylated, and after step 6) TET enzyme-assisted or potassium perruthenate-assisted conversion treatment, its The sequence remains the same, and sequencing it yields genomic DNA information.
  • the template strand is treated with auxiliary reagents, which can convert methylated cytosine into carboxylated cytosine, and then convert carboxylated cytosine into dihydrouracil (that is, two more H atoms) under the action of pyridine borane. Cytosine), dihydrouracil will be recognized as thymine in the sequencing results, and the methylation or/and hydroxymethylation information can be obtained by comparing the sequencing results with the genomic DNA information obtained above.
  • the method further includes: preparing the sequencing library into DNA nanospheres.
  • sequencing can be performed on a DNB sequencer.
  • the method for preparing the DNA nanoball includes: performing rolling circle amplification (Roll circle amplification) on the sequencing library using primer sequences.
  • the primer sequence has a nucleotide sequence as shown in SEQ ID NO: 3 or a core sequence having at least 80% (such as 85%, 90%, 95%, 99%) homology therewith nucleotide sequence.
  • the invention proposes a sequencing library.
  • the sequencing library is obtained by the aforementioned method for constructing a sequencing library. Therefore, using the sequencing library according to the embodiment of the present invention for sequencing, the method and system for sequencing the whole genome DNA and the methylation/hydroxymethylation of the whole genome DNA can be performed simultaneously, and the simultaneous sequencing of DNA and DNA methylation is Completed on one molecule, methylation information can be accurately obtained without reference to gene information, greatly improving the accuracy of methylation information.
  • the present invention proposes the application of the aforementioned sequencing library in sequencing. Therefore, using the sequencing library for sequencing, the method and system for sequencing the whole genome DNA and the methylation/hydroxymethylation of the whole genome DNA can be performed simultaneously, and the simultaneous sequencing of DNA and DNA methylation is completed on one molecule , Accurately obtain methylation information without reference to gene information, greatly improving the accuracy of methylation information.
  • the sequencing includes at least one of whole-genome DNA methylation sequencing and hydroxymethylation sequencing, and whole-genome DNA sequencing.
  • the present invention proposes a method for simultaneously performing whole-genome DNA sequencing and whole-genome DNA methylation or/and hydroxymethylation sequencing.
  • the method includes: sequencing the aforementioned sequencing library to obtain sequencing information, the sequencing information including nascent strand information and template strand information, and the nascent strand information is DNA information of the whole gene;
  • the template strand information is compared and analyzed with the nascent strand information to obtain the genome-wide DNA methylation or/and hydroxymethylation information of the template strand. Therefore, the method according to the embodiment of the present invention can obtain methylation modification information without referring to genome information, and can accurately locate the position of the methylation sequence, thereby improving the accuracy of methylation sequencing data comparison.
  • the comparison analysis includes:
  • the position of the guanine in the nascent chain in the sequencing results
  • the base corresponding to the corresponding position of the complementary strand of the template strand is thymine, which is an indication that no methylation occurs at the position
  • the position of the guanine in the nascent strand corresponds to the complementary strand of the template strand corresponding to
  • the base at the position is cytosine, which is an indication that methylation has occurred at that position;
  • cytosines in the nascent chain are unmethylated cytosines and the dumbbell-shaped double-stranded DNA is converted with TET enzyme and pyridine borane, in the sequencing results, in the nascent chain
  • the base corresponding to the position of guanine in the complementary chain of the template strand is thymine, which is an indication of methylation at the position;
  • the position of guanine in the nascent chain corresponds to the template strand
  • the base at the corresponding position of the complementary strand of the above is cytosine, which is an indication that methylation does not occur at the position;
  • the nascent chain When all the cytosines in the nascent chain are unmethylated cytosines and the dumbbell-shaped double-stranded DNA is converted with potassium perruthenate and pyridine borane, in the sequencing results, the nascent The base corresponding to the position of guanine in the chain and the corresponding position of the complementary chain of the template strand is thymine, which is an indication of hydroxymethylation at the position; the base corresponding to the position of guanine in the nascent chain The base at the corresponding position of the complementary strand of the template strand is cytosine, which is an indication that hydroxymethylation has not occurred at the position;
  • the sequence results , the base corresponding to the position of the guanine in the nascent chain corresponding to the position of the complementary chain of the template strand is thymine, which is an indication of methylation at the position; the position of the guanine in the nascent chain The base corresponding to the corresponding position of the complementary strand of the template strand is cytosine, which is an indication that no methylation occurs at the position.
  • the present invention can obtain genome information and genome methylation information at the same time, and can obtain methylation modification and/or hydroxymethylation information of unknown species without referring to genome information;
  • the present invention uses genome position information to accurately locate the methylation or/and hydroxymethylation sequence position, improving the accuracy of methylation and/or hydroxymethylation data comparison;
  • the present invention does not need to go through PCR, and can effectively and uniformly obtain the methylation and/or hydroxymethylation information of the whole genome;
  • the present invention can realize accurate methylation and/or hydroxymethylation modification detection of C/T polymorphic positions.
  • Figure 1 shows a schematic flow diagram of the preparation process of whole genome DNA and whole genome DNA methylation mixed library based on bisulfite conversion treatment according to an embodiment of the present invention
  • Figure 2 shows a schematic diagram of the preparation process of whole-genome DNA and whole-genome DNA methylation mixed library based on TET-assisted or potassium perruthenate-assisted, according to an embodiment of the present invention
  • FIG. 3 shows a schematic structural view of a joint element 1 and a joint element 2 according to an embodiment of the present invention
  • FIG. 4 shows a schematic diagram of information analysis according to an embodiment of the present invention
  • Fig. 5 shows a flowchart of permutation enzyme sequencing according to an embodiment of the present invention.
  • the present invention proposes a method for simultaneously performing whole-genome DNA sequencing and whole-genome DNA methylation sequencing or/and hydroxymethylation, including:
  • Genomic DNA is randomly interrupted to produce 200-500bp fragments, or DNA that has been interrupted such as cfDNA.
  • fragmented DNA molecules are excisioned by mung bean nuclease on the sticky ends to form blunt ends;
  • Phosphorylate the blunt-ended double-stranded DNA at the 5-end add base A to the 3-end to form a sticky-ended double-stranded DNA molecule with phosphoric acid at the 5-end and base A at the 3-end.
  • adapter element 1 Add adapter element 1 to the above molecule.
  • the main function of the adapter is for subsequent chain extension.
  • the adapter sequence can contain one or more sequencing primer sequences or/and molecular tags (UMI, Unique Molecular Identifiers) or/and samples Label sequence (Index Barcode).
  • UMI Unique Molecular Identifiers
  • Index Barcode samples Label sequence
  • the linker is a special bubble linker (Scheme 3a) with non-complementary sequences in the middle and phosphorylated at the 5-terminus.
  • linker element 1 The 5' and 3' ends of linker element 1 are complementary sequences and one of them has a cohesive terminal U base.
  • the U base can be recognized and excised by the subsequent USER enzyme to generate a cut for polymerase excision or displacement and polymerization extension; or the 5' end and 3' end are complementary sequence cuts containing multiple U bases , with a cohesive terminal T base.
  • the U base can be recognized and excised by subsequent USER enzymes, resulting in one or more nicks, which are used for excision or replacement by polymerase and polymerization extension ( Figure 1).
  • the above ligation product forms one or more cuts under the action of USER enzyme
  • the nascent strand is extended at the nick, and the extension is carried out by an enzyme with 5-3 exonuclease activity (such as T4 DNA polymerase) or 5-3 displacement enzyme activity (such as phi29, Bst).
  • the cytosines in the extended dNTP are all methylated or unmethylated cytosines, and all the cytosines in the original DNA template strand are replaced with methylated or unmethylated cytosines.
  • strand forming a mixed DNA double strand of the original template strand and the nascent strand.
  • the mixed double strands formed above are connected to the linker element 2 to obtain a dumbbell-shaped double-stranded DNA library.
  • the linker sequence includes one or more sequencing primer sequences or/and molecular tags (UMI, Unique Molecular Identifiers) or/and sample tag sequences (Index Barcode).
  • UMI Unique Molecular Identifiers
  • Index Barcode sample tag sequences
  • the linker is a special bubble linker (Schematic 2b), with non-complementary sequences in the middle, complementary sequences at the 5' and 3' ends, sticky T/U bases at the 3' end, and phosphorylation at the 5' end .
  • dumbbell-shaped double-stranded DNA undergoes bisulfite or TET enzyme-assisted conversion treatment, potassium perruthenate (KRuO4), beta glycosyltransferase and TET enzyme-assisted conversion treatment, and the unmethylated modified original template strand Cytosine is converted to uracil or methylated cytosine of the original template strand is converted to dihydrouracil (DHU), while all methylated cytosine of the newly generated strand maintains the same sequence.
  • KRuO4 potassium perruthenate
  • DHU dihydrouracil
  • the transformed dumbbell-shaped double-stranded DNA library is prepared with DNA nanospheres under the action of universal primers.
  • the universal primer is combined with the adapter sequence of the dumbbell-shaped double-stranded DNA, and is linearly extended under the action of an enzyme with displacement activity to generate DNA nanospheres.
  • the DNA nanospheres are loaded onto the DNB sequencing chip for sequencing.
  • the sequencing reaction is carried out. Under the action of the sequencing primers of Read1 and Read2 and the sequencing enzyme with displacement activity (see Figure 3), the original template strand (bisulfite conversion strand, enzyme-assisted or high ruthenium) is respectively measured. Potassium perruthenate (KRuO 4 )-assisted conversion strand) and nascent strand, in which the nascent strand obtains reference genomic DNA information, and the original template strand (bisulfite converted strand, enzyme-assisted, potassium perruthenate (KRuO 4 )-assisted conversion chain) to obtain cytosine conversion information.
  • KRuO 4 Potassium perruthenate
  • KRuO 4 potassium perruthenate
  • a DNB nanopore generates two read lengths Read1 and Read2, of which Read1 or Read2 is derived from the newly generated chain information, and the read is compared to the genome by any comparison software to obtain accurate position information on the genome; the corresponding Read2 Or Read1 is derived from the original template strand (bisulfite conversion strand or enzyme-assisted or potassium perruthenate (KRuO 4 )-assisted conversion strand), compare Read1 and Read2, under the bisulfite conversion condition, the original The position where cytosine is converted to adenine in the template strand is determined to be unmethylated cytosine, and the cytosine that is not converted to adenine is methylated.
  • the main band is about 300bp;
  • the end repair reaction system and conditions are as follows.
  • methylated adapters sometimes also called “methylated tag adapters”.
  • Linker 1 5'-/Phos/GCTCGCAGTCGA GGTCAAGCGGTCTTAGGCTC BBBBBBBB TCTGA AGGACATGGCTA CGATCGACTGCGAGCT-3' (SEQ ID NO: 1)
  • the underlined cytosine is methylated modified cytosine (m5c-dCTP), and B is the sample tag sequence.
  • Linker 2 5'-/5Phos/CGGACTCGACCT GACAATGCATGGCATCTC AGGTCGAGTCCGT-3' (SEQ ID NO: 2) The underlined cytosines in linker 2 are protected by methylation modification
  • CT Conversion Reagent Prepares the CT conversion reagent (CT Conversion Reagent) solution: take out the CT conversion reagent (solid mixture) from the kit, add 900 ⁇ L of water, 50 ⁇ L of M-dissolving buffer (M-Dissolving Buffer) and 300 ⁇ L of M- Dilution Buffer (M-Dilution Buffer), dissolve at room temperature and shake for 10 minutes or shake on a shaker for 10 minutes.
  • M-Dissolving Buffer M-dissolving Buffer
  • M-Dilution Buffer M-Dilution Buffer
  • DNB was quantified using HS Qubit ssDNA kit.
  • the obtained library is subjected to high-throughput sequencing, the sequencing platform MGISEQ-2000, the sequencing type PE100, and the sequenced data are compared and then the basic parameters are counted, including off-machine data, available data, comparison data, etc.
  • the conventional method adopts BS-MAP software to compare, and the method of the present invention adopts BWA software to compare the genome of the nascent chain (cytosine conversion chain) to obtain the accurate position of the read, and then obtain the original template chain (bisulfite) according to the genome comparison position. Conversion chain or enzyme conversion chain) information, and then get accurate methylation alignment information.
  • Using the method of the present invention can greatly improve the methylation comparison rate, and can provide CpG site coverage, can improve the utilization rate of data, and improve the accuracy of methylation detection.
  • the sequencing type is PE100, and the sequencing depth is 30 ⁇ .
  • Data analysis including performance such as data utilization rate, comparison rate, and preference.
  • the main band is about 300bp;
  • the end repair reaction system and conditions are as follows.
  • methylated adapters sometimes also called “methylated tag adapters”.
  • Linker 1 5'-/5Phos/GCTCGCAGTCGAGGTCAAGCGGTCTTAGGCTCBBBBBBBBBTCTGAAGGACATGGCTACGATCGACTGCGAGCT-3' (SEQ ID NO: 1), B is the sample tag sequence
  • Linker 2 5'-/5Phos/CGGACTCGACCTGACAATGCATGGCATCTCAGGTCGAGTCCGT-3' (SEQ ID NO: 2)
  • TET enzyme uses NEBNext Enzymatic Methyl-seq Kit (NEB, E7120S)
  • DNA was purified using PB buffer and Zymo-Spin TM IC Column (Zymo research company), and finally dissolved in 20 ⁇ L TE.
  • DNB was quantified using HS Qubit ssDNA kit.
  • the obtained library is subjected to high-throughput sequencing, the sequencing platform MGISEQ-2000, the sequencing type PE100, the sequenced data are compared and then the basic parameters are counted, including off-machine data, available data, comparison data, etc.
  • the conventional method adopts BS-MAP software to compare, and the method of the present invention adopts BWA software to compare the genome of the nascent chain (cytosine conversion chain) to obtain the accurate position of the read, and then obtain the original template chain (bisulfite) according to the genome comparison position. Conversion chain or enzyme conversion chain) information, and then get accurate methylation alignment information.
  • Using the method of the present invention can greatly improve the methylation comparison rate, and can provide CpG site coverage, can improve the utilization rate of data, and improve the accuracy of methylation detection.

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Abstract

L'invention concerne un procédé de construction d'une banque de séquençage, comprenant les étapes suivantes : 1) fragmentation d'un ADN double brin et soumission des fragments d'ADN obtenus à une réparation de l'extrémité émoussée, à une phosphorylation de l'extrémité 5' et à l'ajout d'une base A à l'extrémité 3' ; 2) ajout respectif du lieur 1 aux deux extrémités de chaque fragment d'ADN obtenu à l'étape 1) au moyen d'une réaction de liaison pour obtenir un produit de liaison ; 3) constitution d'une coupure au niveau d'un site de reconnaissance de l'endonucléase au moyen de l'endonucléase ; 4) réalisation d'une amplification au niveau de la coupure en utilisant le fragment d'ADN lié à une extrémité, qui n'est pas une extrémité collante, de l'élément de liaison 1 comme matrice pour constituer un ADN double brin hybride contenant un brin matrice et un brin naissant ; 5) ajout d'un lieur 2 à une extrémité, non liée au lieur 1, de l'ADN double brin hybride au moyen d'une réaction de liaison pour obtenir un ADN double brin en forme d'haltère ; et 6) soumission de l'ADN double brin en forme d'haltère à un traitement au bisulfite ou à un traitement de transformation pour obtenir la banque de séquençage.
PCT/CN2022/074093 2022-01-26 2022-01-26 Procédé pour effectuer simultanément un séquençage d'adn de génome entier et une méthylation d'adn de génome entier et/ou un séquençage d'hydroxyméthylation Ceased WO2023141829A1 (fr)

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PCT/CN2022/074093 WO2023141829A1 (fr) 2022-01-26 2022-01-26 Procédé pour effectuer simultanément un séquençage d'adn de génome entier et une méthylation d'adn de génome entier et/ou un séquençage d'hydroxyméthylation
CN202280052323.8A CN118076734A (zh) 2022-01-26 2022-01-26 同时进行全基因组dna测序和全基因组dna甲基化或/和羟甲基化测序的方法

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PCT/CN2022/074093 WO2023141829A1 (fr) 2022-01-26 2022-01-26 Procédé pour effectuer simultanément un séquençage d'adn de génome entier et une méthylation d'adn de génome entier et/ou un séquençage d'hydroxyméthylation

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WO2025076733A1 (fr) * 2023-10-11 2025-04-17 深圳华大智造科技股份有限公司 Procédé de construction rapide d'une banque d'acides nucléiques

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WO2012019320A1 (fr) * 2010-08-11 2012-02-16 中国科学院心理研究所 Procédé de séquençage à haut débit de l'adn méthylé et son utilisation
WO2016058134A1 (fr) * 2014-10-14 2016-04-21 深圳华大基因科技有限公司 Élément de liaison et son procédé d'utilisation pour construire une banque de séquençage
WO2016082130A1 (fr) * 2014-11-26 2016-06-02 深圳华大基因研究院 Procédé et réactif pour la construction d'une banque d'acide nucléique simple brin circulaire à double séquence de liaison
CN107586835A (zh) * 2017-10-19 2018-01-16 东南大学 一种基于单链接头的下一代测序文库的构建方法及其应用
CN113337501A (zh) * 2021-08-06 2021-09-03 北京橡鑫生物科技有限公司 一种发卡型接头及其在双端index建库中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012019320A1 (fr) * 2010-08-11 2012-02-16 中国科学院心理研究所 Procédé de séquençage à haut débit de l'adn méthylé et son utilisation
WO2016058134A1 (fr) * 2014-10-14 2016-04-21 深圳华大基因科技有限公司 Élément de liaison et son procédé d'utilisation pour construire une banque de séquençage
WO2016082130A1 (fr) * 2014-11-26 2016-06-02 深圳华大基因研究院 Procédé et réactif pour la construction d'une banque d'acide nucléique simple brin circulaire à double séquence de liaison
CN107586835A (zh) * 2017-10-19 2018-01-16 东南大学 一种基于单链接头的下一代测序文库的构建方法及其应用
CN113337501A (zh) * 2021-08-06 2021-09-03 北京橡鑫生物科技有限公司 一种发卡型接头及其在双端index建库中的应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025076733A1 (fr) * 2023-10-11 2025-04-17 深圳华大智造科技股份有限公司 Procédé de construction rapide d'une banque d'acides nucléiques

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